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Unofficial guide to assessing your RNA

Below is our internal comparison of various RNA kit/assays for assessing viral RNA quantity and quality

RNA quantity and quality can greatly affect your RT-qPCR or RNAseq data downstream so it is important to assess it appropriately.

A perfect RNA assay would have:

  1. high sensitivity,
  2. high accuracy,
  3. high selectivity,
  4. have a large linear range,
  5. and be easy to run.

None of the kits/assays we have tested fulfill all of these ideals, thus, we set out to make recommendations for various situations.

Important things to assess RNA quality for your specific assay

  1. Contamination (phenol etc): Pure RNA 260/280 ratio is ~2.0. Some downstream assays/enzymes are more sensitive than others
  2. Degradation/Quality: Generally quality RNA RIN value is greater than/equal to 7 or 8 for sequencing. PCR may be more lenient unless your amplicon target is very large.
  3. Concentration: sequencing is very sensitive to accurate assessment of the concentration of only the usable RNA (excluding background contaminates) while PCR is not as long as there is enough starting sample material. NOTE: Incredibly low amounts of RNA is probably too low for any accurate assessment and the result will be more an estimate.
  • Once you have determined which of the above 3 quality metrics is most important to your assay/enzyme, refer to the table below for our recommendation for associated RNA assessment assay

Experiment: Two different techs were given the same 4 viral RNA samples diluted from ~0.5-1 ng/uL and~50-100 ng/uL to test across these assays: NanoQuant (NanoDrop™), Agilent Bioanalyzer 2100 RNA 6000 Nano, Quant-iT™ RiboGreen® RNA, and Qubit® RNA HS Assay.

Vironomics Core in-house viral RNA comparison

 

Concentration (within range)

 

NanoQuant/NanoDrop Agilent RNA 6000 Nano (2100)
 Quant-iT RiboGreen (high range) Qubit RNA HS Assay
Recommendation
  • Contamination
  • Concentration for very low samples
 Quality Concentration (within range)
Approx. linear range (tested to 100ng/uL) ~1 ng/uL-100 ng/uL Not linear ~5 ng/uL-100ng/uL♦ ~5 ng/uL-100 ng/uL
Approx. limit of detection ~1 ng/uL ~25 ng/uL ~5 ng/uL♦ ~5 ng/uL♠
Approx. precision (R2) ~0.965 ~0.856 ~0.992 ~0.993
Known Issues It will continue to provide concentration values, even when below the limit of detection and inaccurate♥ It will continue to provide concentration values, even when below the limit of detection and inaccurate♥ It will continue to provide concentration values, even when below the limit of detection and inaccurate♥

♦Testing the low range could result in a lower limit of detection (~1 ng/uL expected).
♠ Increasing the input volume could give accuracy with lower concentration of sample.
♥ While this problem is obvious when testing dilutions and replicates, it is difficult when you are just testing a single sample. For Agilent and RiboGreen, dilutions below the limit of detection continued to read approximately as high as the previous dilutions. It would therefore be difficult to determine whether an individual sample reading 5 ng/uL sample is at 5 ng/uL or is actually much lower and reading inaccurately.